Surface-enhanced Raman spectroscopy (SERS) is a spectroscopic technique, where Raman scattering is boosted primarily by enhanced electric field due to localized surface plasmon resonance (LSPR). With advances in nanofabrication techniques, SERS has attracted great attention for label-free molecular sensing and imaging. However, the practical use of SERS has often encountered a couple of inherent issues. The first one is regarding a molecule transfer step where target molecules need to be within the close proximity of a SERS-active surface by either mixing with nanoparticles or coating onto surface-bound nanostructures. In other words, target molecules are required to be transferred from non-SERS-active surfaces to SERS-active ones, normally in the solution phase, which can be problematic due to issues such as surface affinity variability and uncertainty, competitive adsorption among different molecules, and contamination issues, causing irreproducible results and erroneous or biased interpretations. More importantly, if the spatial distribution of molecules on the surface prior to the transfer step is of importance, such information is completely lost. Practically, solution-phase processes are relatively more labor and time-consuming and require a “wet” laboratory. Furthermore, SERS measurements are always restricted to molecules adsorbed on metals such as Ag, Au, and Cu.
To address the aforementioned issues, many approaches have been developed, and can be broadly classified into chemical and physical means. Chemical approaches employ functionalized surfaces to improve affinity and selectivity of target molecules. For example, Au nanoparticles were surface-modified by cystamine and cysteine for detecting perchlorate and trinitrotoluene, respectively. Physical approaches, in contrast, attempt to bring the target molecules to the SERS-active surface by physical manipulation. A potential advantage of physical approach lies in that the SERS enhancement depends solely on distance, rather than surface affinity. For example, tip-enhanced Raman scattering (TERS) technique introduces enhanced electromagnetic field by bringing a nano-tip into the vicinity of target molecules. Although TERS provides diffraction-unlimited spatial resolution similar to that from atomic force microscope, it is time-consuming for large area imaging. Li et al. developed shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS), in which SERS-active nanoparticles are coated over the surface with target molecules via wet processes. Lee et al. demonstrated using gold nanorod-loaded filter paper for SERS measurement by pre-wetting and swabbing it on the surface to be probed.
SERS analyses have been performed to analyze biological samples. For example, urine is an easily-accessible bodily fluid that provides metabolic information, including the renal status. Creatinine, a protein metabolite, is one of the major components of human urine besides urea. Since the content of creatinine excreted into the urine is relatively constant in the absence of renal disease, it is used as an internal standard to normalize variations in other urine analytes. Moreover, the detection of creatinine concentration in the urine is important for renal clearance tests, which monitor the filtration function of the kidney. Serum creatinine concentrations are routinely monitored as an indicator of clinical renal function. In clinical diagnostics, serum and urine creatinine concentrations are measured, and generally a high creatinine level indicates kidney problems. For example, normal levels of blood creatinine are approximately 0.6 to 1.2 mg/dl in adult males and 0.5 to 1.1 mg/dl in adult females. In urine, however, creatinine is found to be 500-2000 mg over a 24-hour period. By simply dividing 500-2000 mg to the average urine volume of 1-2 L in an adult male, the lower limit of urine creatinine concentration is estimated to be ˜25 mg/dL.
Due to the importance of creatinine in clinical research, a variety of analytical methods have been developed for detecting creatinine in urine, including Jaffe reaction spectrophotometric method, enzymatic method, capillary zone electrophoresis, high performance liquid chromatography (HPLC), high performance thin-layer chromatography (HPTLC), liquid chromatography tandem mass spectrometry (LC-MS), gas chromatography mass spectrometry (GC-MS), isotope dilution extractive electrospray ionization tandem mass spectrometry (ESI-MS), Raman spectroscopy and surface-enhanced Raman scattering (SERS). Compared to traditional analytical methods, Raman and SERS methods offer several advantages. They require no reagents or separation, are non-invasive, are capable of qualitative and quantitative measurements, and provide molecular structure information. In particular, SERS is a highly-sensitive Raman spectroscopic technique where Raman scattering is enhanced primarily by near-field electromagnetic enhancement due to localized surface plasmon resonance (LSPR). Recent advances in the field of nanotechnology have paved the way for the development of SERS based detection.
Most creatinine SERS analyses have thus far been performed on metallic (e.g., Ag and Au) colloidal nanoparticles. In general, Au-based SERS substrates are more stable, nontoxic and biocompatible compared to Ag-based ones, although they have inherently lower (i.e. 102-103 fold) SERS effects than Ag-based substrates. By using gold colloids, the potential of SERS for qualitative and quantitative creatinine measurements was illustrated by W. R. Premasiri et al., and the measurement of creatinine in human urine at concentrations ranging from 2.56 to 115.2 mg/dl was reported by T. L. Wang et al. Y. Wang et al. performed the detection of creatinine water solution with concentrations ranging from 10-280 mg/dl by mixing with silver colloids. R. Stosch et al. described the determination of creatinine in human serum at physiologically relevant levels using silver colloids as SERS substrates. In addition, nanostructured metal surfaces have been employed for SERS measurements. Compared to metallic colloids, a significant advantage of nanostructure based approach is that SERS signals are more stable against sample ionic strength. This is because the ionic strength can affect the aggregation of metallic colloids and adversely influence reproducibility. H. Wang et al. conducted quantitative analysis of creatinine in the urine of healthy and diabetic patients using Ag-coated parylene nanostructures as the SERS substrate, and successfully detected as low as 6.1 mg/dl urine creatinine. K. W. Kho et al. analyzed urine samples in a microfluidic device embedded with Au-coated polystyrene nanosphere arrays as the SERS substrate. Among existing reports, the lowest detectable concentration was 0.1 μg/ml (˜0.88 μM) in water, and 2.56 mg/dl in real urine samples.
Although SERS has the potential for creatinine sensing, both the limit of detection and reproducibility need further improvement for practical application. As mentioned previously, colloidal SERS substrates suffer from sample ionic strength dependent aggregation. Planar nanostructures, on the other hand, may not provide low enough detection limit, because the surface area within the source laser footprint is small and the light-matter interaction is limited. This calls for the development of robust, uniform, and reproducible SERS substrates and reliable measurement techniques.